Pestic. Sci. 1975, 6, 395-403

Persistence of Gamma-BHC and Beta-BHC in Indian Rice Soils under Flooded Conditions

Rangappa Siddaramappa and Nambrattil Sethunathan

Laboratory of Soil Microbiology, Central Rice Research Institute, Cuttack-6, Orissa, India

(Manuscript received I2 February 1975 and accepted 24 April 1975)

The relative persistence of [14C]-gamma-BHC and [W]-beta-BHC in Indian rice soils under flooded conditions was studied. In alluvial, laterite and pokkali (acid sulphate, saline) soils, rapid degradation of both isomers occurred; in sandy and kari (acid sulphate, saline) soils, both isomers persisted even after 41 days of flooding. The rapid degradation of BHC isomers in the former three soils was related to highly negative redox potentials within 20 days of flooding in contrast to oxidised conditions in sandy and kari soils even after 41 days. During the degradation in the soils, beta-BHC showed longer lag than gamma-BHC. Results suggest that the degradation of beta-BHC commences at a potential lower than that required for gamma-BHC degradation. Greater decomposition of gamma-BHC occurred in rice straw-amended soils than in unamended soils when the insecticide was incorporated to the soils in an aqueous solution. Addition of BHC isomers to the soils in ethanol resulted in comparable rates of rapid de- composition in both rice straw-amended and unamended soils, since ethand was as effective as rice straw in lowering the redox potentials of the soils favouring BHC decomposition in unamended soil as well.

1. Introduction

Chlorinated hydrocarbon insecticides are known to be both chemically and biologically more stable in the environment than organophosphorus and carbamate insecticides. However, recent investigations have revealed that certain chlorinated hydrocarbons such as 1,2,3,4,5,6-hexachlorocyclohexane (BHC), DDT and methoxychlor are biodegradable in a predominantly anaerobic ecosystem such as flooded soil.1

Commercial preparations of BHC generally contain gamma-, beta-, alpha- and delta-isomers of which gamma-isomer is the most insecticidal and beta-isomer is the most persistent. Entry of beta-BHC into the food chain after application of commercial BHC to crops has caused concern in Japan.2 33 According to Kawahara et d4 beta- BHC appeared to be the most persistent isomer in Japanese rice soils, followed by alpha-, gamma-, and delta-isomers. However, MacRae et aZ.5 reported that beta-BHC was as biodegradable as gamma-BHC in two tropical Philippine soils under flooded conditions. Although these reports show that BHC isomers are decomposed rapidly in flooded soils, the quantity and identity of degradation products have not been established.6 Decomposition of pesticides in saline environments has not received

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396 R. Siddaramappa and N. Sethunatban

much attention except perhaps for the reports on the persistence of parathion in two saline acid sulphate rice soils7 and of DDT in saline water.8 In view of the extensive use of BHC in Indian agriculture, particularly in the control of rice pests, the fate of gamma-BHC and beta-BHC in Indian rice soils including two unique saline rice soils, characterised as acid sulphate soils, under flooded conditions was investigated.

2. Materials and methods 2.1. Soils Some of the characteristics of the five soils used in this study are listed in Table 1. Among the soils used, two saline acid sulphate soils from Kerala, South India, are unique rice soils of extreme acidity and are locally known as kari and pokkali soils.9

Table 1. Chemical characteristics of the soils

Acid sulphate Acid sulphate Characteristics (Kari) Alluvial (Pokkali) Sandy Laterite

PHa 3 .O 6 . 2 4 .2 6 . 0 5 . 0 Organic matter (%) 27.81 1.61 8 .21 0.02 3 .25 Electrical conductivity 15.0 0 . 6 8 . 5 0 . 4 0 .2 (mmhos cm-1)"

a Measured on 1 : 1.25 soil-water suspension.

Techn. Workstatten, Weilheim, Obb. Germany). Electrical conductivity of saturation extract of the soil determined in conductivity meter (Wiss.

2.2. Labelled compounds Uniformly labelled [l'Cj-garnma-BHC (specific activity, 45 Ci/mol) and [14C]-beta- BHC (specific activity, 36.1 Ci/mol) were obtained from the Radiochemical Centre, Amersham, England. The labelled BHC isomers were dissolved in hexane (100 ml) after evaporating off the benzene carrier. An aliquot (20 ml) of this stock solution was evaporated to dryness and the residues were redissolved in ethanol prior to incorpor- ation with the soils. In one experiment on the effect of rice straw on gamma-BHC, the residues were equilibrated with distilled water (120 ml) for 24 h for incorporation in aqueous form. Purity of labelled BHC isomers was confirmed by thin-layer chromato- graphy (t.1.c.).

2.3. Soil incubation studies The soils (20 g) were placed in test tubes (200 x 25 mm diam.). Labelled BHC isomers were introduced to the soils in ethanol (0.1 ml). After 2 h, commercial 5 % BHC granules (5 mg) were added to the soils as a carrier. The soils were then flooded with distilled water (25 ml) and incubated at room temperature (28 4 "C). At intervals, two replicate tubes were removed for analysis.

Persistence of BHC in rice soils 397

2.4. Effect of rice straw Only alluvial soil from the institute farm was used in this study. The soil (20 g) was thoroughly mixed with rice straw powder at 0 .5 % (w/w) level in test tubes (200 x 25 mm diam.). In the first experiment on the effect of rice straw on the persistence of gamma-BHC, an aqueous solution (5 ml) of [14C]-gamma-BHC was added to rice straw-amended and unamended soils and the soils were flooded after 2 h with an aqueous solution of non-labelled gamma-BHC (20 ml) (obtained from National Physical Laboratory, Chemical Standard Division, Middlesex, England), as a carrier. Two replicate tubes for each treatment were removed for residue analysis. In the second experiment on the effect of rice straw on the relative persistence of gamma- and beta-BHC in flooded alluvial soil, the labelled isomers were introduced to the soils in ethanol (0.1 ml) and not in aqueous form and after 2 h, the soils were flooded withdistilled water (25 ml). At intervals, two replicate tubes were removed for analysis.

2.5. Extraction and residue analysis Residues in the soils were extracted with chloroform-diethyl ether (1 : 1) three times as described earlier for parathion.lo The solvent extract was evaporated to dryness at room temperature. Residues were redissolved in methanol. An aliquot of this solution was added to 5 ml of liquid scintillator NE 213 (Nuclear Enterprises Ltd, Sighthill, Edinburgh, Scotland) which consists of 2,5-diphenyloxazole (PPO) ( 5 g), 1,4-di- [2-(5-phenyloxazolyl)]benzene (POPOP) (0.3g) and toluene (1 litre) and the total radio- activity determined in a Liquid Scintillation Counter Model LSS 20 (Electronics Corporation of India Ltd, Hyderabad, India). For quantitative determination of parent compounds, the residues dissolved in methanol were separated by t.1.c. together with authentic compounds, employing acetone-hexane (5 : 95) as developing solvent. The authentic compounds were located by spraying with an ethanolic solution of 0.085 % silver nitrate and 2.5 % ammonium hydroxide and subsequent exposure to ultraviolet light. The silica gel areas of the samples in the chromatoplate opposite the authentic compounds were scraped off carefully and transferred to test tubes. Residues were eluted in diethyl ether. An aliquot was added to the scintillator solution (5 ml) and the radioactivity determined.

In one experiment on the effect of rice straw ongamma-BHC, aproportional counting system was employed instead of a liquid scintillation system. For total radioactivity, residues dissolved in methanol were placed in planchets and the radioactivity was determined, immediately after evaporation at room temperature, employing a gas-flow proportional counter (Electronics Corporation of India Ltd, Hyderabad, India). For quantitative determination of the parent compound, residues from silica gel areas were eluted in diethyl ether as described earlier, an aliquot of this solution was evaporated in a planchet and the radioactivity determined in a proportional counter.

2.6. Radioautograph The thin-layer chromatograms of BHC residues were exposed to Kodak X-ray no screen film for 25-30 days in a Siemens metal casette.

398

2.7. Redox potential (Eh) and pH measurements For Eh determination, soil samples (50 g) after appropriate amendments were flooded with distilled water (62 .5 ml) in a 100 ml beaker to provide the same soil-water ratio as used in the samples contained in test tubes. E h was measured with a portable redox meter Model RM-IF (TOA Electronics Ltd, Tokyo, Japan) fitted with a compound platinum and calomel electrode type GC-21 I . After Eh measurements, the pH of the soil samples was determined in an Elico pH meter Model L1-10.

R. Siddaramappa and N. Sethunathan

3. Results and discussion 3.1. Persistence of gamina-BHC and beta-BHC in different soils Persistence of gamma-BHC and beta-BHC in five Indian soils under flooded conditions was studied employing an isotope technique. The total radioactivity in the solvent extracts of the soils treated with gamma-BHC and beta-BHC decreased with incubation in the alluvial, laterite and pokkali soils without any appreciable increase in that of the water phase, whereas in kari and sandy soils no appreciable decrease in the radio- activity occurred (Table 2) . Determination of radioactivity of the parent compounds after t.1.c. separation revealed that in agreement with total radioactivity, the most rapid degradation of both these BHC isomers occurred in the alluvial soil followed by

Table 2. [14C]-Gamma-BHC and [I4C]-beta-BHC degradation in soils under flooded conditions

Incubation ~ - (days) Kari

[ W]-Gamma-BHCa 0 43.3 (45.1)

20 31.5 (32 0) 41 24.8 (25 0)

0 49.6 (50.7) 20 42.0 (42.4) 41 36.0 (36.7)

[14C]-Beta-BHCa

14C recovered (10-4 counts/min/20 g soil) ~ - ~ ~

Alluvial Pokkali Sandy Laterite ~ - - ~

51.7 (57.5) 44.9 (49.9) 49.4 (55.6) 52.2 (59.1) 2.3 (12.4) 25.3 (31.3) 47.2 (52.9) 19.4(28.2) 0.8 (2.9) 2.1 (8.6) 42.4(44.4) 1.3 (8.2)

71.8(75.9) 63.4(66.1) 79.9(82.7) 75.6(81.6) 16.2 (25.1) 58.9 (62.0) 73.5 (79.8) 45.2 (62.3) 1.9 (4.3) 20.7 (23.1) 65.5 (70.0) 2.9 (11.4)

a [14C]-Gamma-BHC and [14C]-beta-BHC recovered by t.1.c. from the spots with RF values identical to analytical grade reference compounds. Figures in parenthesis represent total radioactivity recovered in the chloroform-diethyl ether phase.

laterite and pokkali soils; in kari and sandy soils, both gamma-BHC and beta-BHC persisted. Gamma-BHC decreased more in 20 days than did beta-BHC in alluvial, laterite and pokkali soils; in pokkali soil particularly no appreciable loss of beta-BHC was noticed after 20 days in contrast to a more than 50 % declinein gamma-BHC levels at this time. Measurements revealed that the lower the value of Eh the greater was the degradation of both these BHC isomers. The Eh reached negative values within 20 days of flooding in alluvial, laterite and pokkali soils whereas in kari and sandy soils, in which no degradation occurred, the Eh was positive even at 41 days after flooding

Persistence of BHC in rice soils 399

Table 3. pH and E h changes in soils treated with gamma-BHC and beta-BHC following flooding

Alluvial

PH Eh (mV)

Pokkali

pH Eh (mV)

Sandy

pH Eh(mV)

[14C]-Gamma-BHCa 0 3.0 +390

20 4.0 +220 41 4.2 +225

0 3.0 +380 20 4.0 +160 41 4.2 +165

[14C]-Beta-BHCa

6.2 +235 7.0 -140 7.1 -140

6.2 +255 7.1 -120 7.1 -145

4.2 +250 6.4 -100 6.7 -115

4.2 +250 6.3 -80 6.7 -100

Laterite

6.1 +200 6.5 +125 6.6 +SO

6.0 +220 6.5 +150 6.8 +45

5.0 +240 6.4 -80 6.6 -70

5 . 1 +255 6.5 -50 6.7 -75

a Gamma-BHC and beta-BHC were incorporated to the soils in ethanol.

(Table 3). At 20 days, the average Eh and pH of pokkali soils were - 90 mV and 6.35 respectively and at 41 days, the corresponding values were - 110 mV and 6.7. The rapid decrease in beta-BHC levels after a lag of at least 20 days, therefore, indicated that potentials of about - 80 mV would favour its rapid loss from the soils. In the two acid sulphate saline soils used, more rapid degradation of BHC isomers in pokkali soil than in kari soil appeared to be due to lower potentials and a near neutral pH in the former soil after 20 days of flooding apparently providing an optimum condition for the growth of anaerobic microorganisms. Earlier reports show that anaerobic bacteria are involved in the rapid disappearance of gamma-BHC from flooded soils.llJ2 The negative relationship between Eh and degradation of BHC isomers in different soils used in this study suggest the role of anaerobic microorganisms in the degradation of BHC isomers.

Radioautography of the residues in the solvent extracts of the soils after t.1.c. separation revealed a prominent spot of gamma-BHC (RF, 0.65) and a faint spot (RF, 0.93) in all the soils treated with gamma-BHC (Figure 1). The minor product (RF, 0.93) was also detected in the radioautograph of gamma-BHC residues extracted immediately after its application to soils which were previously autoclaved three times on alternate days for 1 h each at 15 lb/in2 treated with gamma-BHC. In addition, this minor product was formed when gamma-BHC in aqueous solution (soil-free) was extracted with the same procedure, indicating that this minor compound was a product of chemical reaction formed during extraction. Similarly, radioautograph after t.1.c. of beta-BHC residues in the soils revealed a prominent spot of beta-BHC (RF, 0.50) and a faint spot (RF, 0.94). It is probable that the minor products from gamma-BHC and beta-BHC may be the respective isomers of pentachlorocyclohexane (PCCH), apparently formed by chemical reaction. No attempt was made, however, to identify the minor products. No other radioactive compound was detected in the radioautographs of gamma-BHC and beta-BHC residues irrespective of the soil types although gamma-3,4,5,6-tetrachlorocyclohex-l-ene (gamma-BTC) was detected in Japanese rice soils treated with gamma-BHC under flooded conditions, employing gas-liquid chromatography.13 According to Tsukano and Kobayashi,l3 gamma-BTC disappeared much faster than gamma-BHC. Yule er all4 reported the formation of

400 R. Siddaramappa and N. Sethunathan

Figure 1. Radioautograph of gamma-BHC (a) and its breakdown product (b) formed during extraction with chloroform-diethyl ether (1 : 1) immediately following application of gamma-BHC to the soil.

gamma-PCCH from gamma-BHC by a biological action in moist Canadian soils. Formation of such unstable metabolites in the soils could not be ruled out although no intermediates were detected in the extraction and analytical methods used in our study.

3.2. Effect of rice straw The effect of rice straw on the persistence of gamma-BHC in an alluvial soil under flooded conditions was investigated. The insecticide was applied to the soils in an aqueous solution. The radioactivity recovered in the solvent phase and as parent compound after t.1.c. separation, Eh and pH changes are given in Table 4. No appreci- able degradation of gamma-BHC occurred in unamended soils until 20 days whereas in rice straw-amended soils, the insecticide concentration decreased rapidly at 7 days and reached low levels in 20 days. However, rapid loss of insecticide from unamended soils occurred between 20 and 30 days. The total radioactivity in the solvent phase followed the same trend as the parent compound. The enhanced gamma-BHC degradation in rice straw-amended soils was associated with lower redox potentials of the amended soils compared to those of the unamended soils. In amended soils, Eh dropped from +205 mV to -40 mV in 1 1 days and to - 160 mV in 20 days; in unamended soils, Eh values were + 90 mV at 11 days and - 90 mV at 20 days. Addition of organic matter to flooded soils is known to enhance the reduction of the soil components to lower states of oxidation as a result of increased microbial respiration.15 Evidently, rice straw lowers the Eh of the flooded soils to a level favour- able for the anaerobic/facultatively anaerobic microorganisms involved in gamma-BHC degradation. Yoshida and Castrole also reported more rapid degradation of gamma- BHC in rice straw-amended than in unamended soils.

Persistence of BHC in rice soils 401

Table 4. Effect of rice straw on gamma-BHC degradation in flooded alluvial soila

Rice straw-amended soil Unamended soil ___ _- _ _ _ ~

Radioactivity recoveredb Radioactivity recoveredb

(days) pH (mV) TotalC Gamma-BHCd pH (mV) Totalc Gamma-BHC" E h Eh ~--_______ Incubation

______ 0 6 2 t-205 3 5 2 3 6 2 +220 2 8 1 .8 3 6 4 +135 3.5 2 0 6 4 +160 3.7 2 .2 7 6 8 - 5 1 6 1 0 6 5 +110 2 4 2 1

11 6 9 -40 1 6 0 8 6.7 +90 2 6 2 1 20 7 0 -160 0 3 0.1 6.9 -90 2.1 2 0 30 6 9 -160 0 1 0 0 6.9 -100 0.7 0 4

~~

a Gamma-BHC was added to the soils in an aqueous solution. * l4C recovered in c Total radioactivity in the chloroform-diethyl ether phase. d [14C]-Gamma-BHC recovered by t.1.c. from the spots with RF values identical to analytical

counts/min/20 g soil as determined in gas-flow proportional counter.

grade reference compound.

In the next experiment on the effect of rice straw on the relative persistence of gamma-BHC and beta-BHC in the flooded alluvial soil, BHC isomers were applied to the soils in ethanol. In contrast to the more rapid decomposition of gamma-BHC in amended soil than in unamended soil in the earlier experiment (Table 4), gamma- BHC levels decreased rapidly in both amended and unamended soils and the degradation rates were comparable when the insecticide was added to the soils in ethanol (Table 5) . An explanation for this behaviour was provided by Eh measure- ments. Eh changes were essentially the same leading to negative values in both amended

Table 5. Effect of rice straw on gamma-BHC and beta-BHC degradation in flooded soil ~~

Rice straw-amended Unamended ______ ____ Incubation --

(days) pH Eh (mv) l4c recoveredb pH Eh (my) 14C recovered0 - - -__ --____ _ _ _ _

[14C]-Gamma-BHCa 0 6 2 f 2 1 5 51.OC(78 3) 6 2 +200 42.lC(71.5) 7 6.9 - 50 24 2 (54.7) 7 0 - 15 30 2 (56.4)

14 6.9 - 35 12.7 (24 4) 7 2 - 40 16 3 (30.7)

$0 7 0 -155 0.3 ( 5 . 8 ) 7.1 -165 0 3 ( 4.2)

0 6.2 f 2 0 0 84.9c(89.3) 6 2 +200 77.6" (78.9) 7 6.9 - 50 70.8 (71 8) 7 0 - 30 68.6 (69.3)

14 6.9 -40 67 2 (72 8) 7.1 - 40 69.4 (73.3) 25 6.9 -160 7 5 (12 0) 7.0 -155 18 0 (24.0) 40 7.0 -160 9 2 (14.8) 7 0 -120 9.9 ( 1 1 . 3 )

25 6.9 -160 6 0 (15 7) 7 0 -160 1.2 ( 7.9)

[14C]-Beta-BHCa

Q BHC isomers were applied to the soils in ethanol.

c [%]-Gamma-BHC and beta-BHC recovered by t.1.c. from the spots with RF values identical to analytical grade reference compounds. Figures in parenthesis represent total radioactivity recovered in the chloroform-diethyl ether phase.

1% recovered in counts/min/20 g soil.

5

402 R. Siddaramappa and N. Sethunathan

and unamended soils within 7 days. Evidently, ethanol used for incorporating gamma- BHC appeared to act as an energy substrate for microbial activity resulting in a rapid drop in E h of unamended soils as well. Ethanol was as effective as rice straw in lowering the E h of the flooded soils and rice straw did not have any additive effect on E h in the presence of ethanol.

In several reported studies on persistence, the pesticides are often incorporated into the soils in a solvent such as ethanol or acetone because of their extreme insolubility in water. If, as in most cases, the degradation is microbiological, we could expect enhanced degradation of water-insoluble organic compounds if introduced in organic solvents. Gunner et a1.l' reported that diazinon was rapidly decomposed by bacteria only if introduced into the medium in ethanol. Our studies indicated that under a flooded ecosystem, the ethanol used for incorporating gamma-BHC further accelerated the reduction of the soils favouring the anaerobic decomposition of the insecticide. Hamaker18 pointed out that in sealed jars of limited space used in several studies on pesticide metabolism, anaerobic conditions would be created inadvertently in the moist soils due to the rapid utilisation of oxygen during soil respiration, if proper aeration is not provided. In such a closed system, anaerobiosis would further be enhanced by the presence of ethanol or similar solvents used for dissolving the pesti- cides.

Regarding the effect of rice straw on the relative persistence of gamma-BHC and beta-BHC in flooded alluvial soils, no appreciable degradation of beta-BHC occurred in both amended and unamended soils until 14 days; during the same period, more than 50% of the added gamma-BHC was lost (Table 5) . The E h dropped to -40 mV during this period. Between 14 and 25 days, the Eh of both amended and unamended soils decreased to about - 160 mV and during this period, considerable degradation of beta-BHC occurred with the rate slightly faster in amended soils. The E h decline was essentially the same in both amended and unamended soils due to the added presence of ethanol as discussed earlier. The longer lag in the degradation of beta-BHC as compared to gamma-BHC in this experiment (Table 5) as well as in different soils, particularly pokkali soil, in the earlier experiment (Table 2) appeared to be related to Eh. Considerable degradation of gamma-BHC occurred above a potential of -40 mV whereas degradation of beta-BHC started only when the Eh was lowered below - 40 mV. Although these isomers are known to decompose rapidly in flooded soils,5J6 the relationship between E h of the soils and their persistence in the soils had not been investigated earlier.

4. Conclusions

The data presented in this report reveal that gamma-BHC and beta-BHC decomposed rapidly if the redox potentials of the soils are lowered to a range of -40 to - 100 mV following flooding. Even in a highly saline acid sulphate soil such as pokkali soil, these isomers are degradable because of a rapid dropin& to highlyfavourable negative values. The initial lag followed by a rapid decrease in the concentration of gamma- and beta- isomers of BHC in three soils concomitant with an E h drop to negative values suggested participation of anaerobic microorganisms in their degradation.

Persistence of BHC in rice soils 403

Addition of rice straw or incorporation of BHC isomers in an organic solvent further accelerates the reduction of the flooded soils and thereby the disappearance of BHC isomers. Caution is warranted in interpreting the studies on the persistence of water-insoluble pesticides incorporated in organic solvents since the presence of added organic solvents would inadvertently result in anaerobiosis particularly, in models of sealed containers and flooded soil ecosystems.

Acknowledgements

Research supported in part by a grant from the International Atomic Energy Agency, Vienna, Austria (Contract No. 1179) and from Department of Atomic Energy, Government of India (Contract No. BRNS/F&A/42). The authors are grateful to Dr S . Y. Padmanabhan, Director, for encouragement and facilities and Dr W. H. Freeman, All India Coordinated Rice Improvement Project, Rajendranagar, Hyderabad-30, India, for a gift of the redox meter used in this study.

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Agriculture and Forestry, Tokyo, Japan, 1971, p. 319. 4. Kawahara, T.; Matsui, M.; Nakamura, H. Bull. agric. Chem. Inspection Stn 1972, 12, 42. 5. MacRae, I. C.; Raghu, K.; Castro, T. F. J . agric. Fd Chem. 1967,15,911. 6. Fries, G. F. Adv. Chem. Ser. 1972, 111, 256. 7. Sethunathan, N. J. agric. Fd Chem. 1973, 21, 602. 8. Maddox, J. Nature, Lond. 1972, 236, 433. 9. Bloomfield, C.; Coulter, J. K. Adv. Agron. 1973, 25, 266.

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J. W. Eds), Marcel Dekker, 1972, p. 255.